Directed evolution of hemeproteins for selective cyclizations

• Applicant: Dr. Grzegorz Kubik
• Subject Area: Biological and Biomimetic Chemistry ; Organic Molecular Chemistry - Synthesis and Characterisation
• Term: from 2016 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 313888180

In summary, within this study synthetic tools for the production of a large variety of heterocyclic compounds, which are of high importance in many pharmaceutics, will be created. The principle of this method is to trap intermediate ylides with electrophiles in enzymatic heteroatom-hydrogen bond insertion of carbenoids and nitrenoids. For this, at first the required scope of insertion reactions at heteroatom-hydrogen bonds will be generated by directed evolution of Cytochrome P450. Next the enzyme variants will be further engineered to facilitate the trapping reaction, employing three different electrophilic groups. The benefits of enzymes as catalysts are defined by the high selectivity that is provided frequently and the possibility to reengineer those enzymes to obtain selectivities of choice, for example switching the selectivity from one enantiomer to the other. Furthermore mild and environment friendly reaction conditions can be applied, meaning a sustainable alternative to up to date methods. Within this project also new insights into ylide formation, stability and reactivity may be raised that allow to understand in detail how to switch reactivity. One very interesting aspect is that for some of the trapping reactions a spectroscopic high throughput assay can be set up to track the reaction. This will allow for screening of enzyme libraries containing random mutations throughout the complete amino acid sequence. Till now the investigated P450 libraries mainly focused on mutations at sites contributing to the catalytic pocket due to the lack of such an assay. So within this project completely unknown structure-activity relations within parts of P450 not contributing directly to the catalytic pocket could be disclosed.

• DFG Programme: Research Fellowships
• International Connection: USA
• Host: Professorin Frances H. Arnold, Ph.D.